Jalapinoside, a Macrocyclic Bisdesmoside from the Resin Glycosides

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Jalapinoside, a Macrocyclic Bisdesmoside from the Resin Glycosides of Ipomea purga, as a Modulator of Multidrug Resistance in Human Cancer Cells Elihú Bautista, Mabel Fragoso-Serrano, and Rogelio Pereda-Miranda* Departamento de Farmacia, Facultad de Química, Universidad Nacional Autónoma de México, Mexico City, 04510 DF, Mexico S Supporting Information *

ABSTRACT: The first macrocyclic bisdesmoside resin glycoside, jalapinoside (4), was purified by preparative-scale recycling HPLC from the MeOH-soluble extracts of Ipomoea purga roots, the officinal jalap. Purgic acid C (3), a new glycosidic acid of ipurolic acid, was identified as 3-O-β-D-quinovopyranoside, 11-O-β-D-quinovopyranosyl-(1→2)-O-β-D-glucopyranosyl-(1→3)-O[β-D-fucopyranosyl-(1→4)]-O-α-L-rhamnopyranosyl-(1→2)-O-β-D-glucopyranosyl-(1→2)-O-β-D-quinovopyranoside (3S,11S)dihydroxytetradecanoic acid. The acylating residues of this core were acetic, (+)-(2S)-methylbutanoic, and dodecanoic acids. The site of lactonization was defined as C-3 of the second saccharide moiety. Reversal of multidrug resistance by this noncytotoxic compound was evaluated in vinblastine-resistant human breast carcinoma cells.

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soluble content.2 According to Mannich and Schumann,8 convolvulin resin glycosides are composed as large, high molecular weight polymers of oligosaccharides glycosidically linked to a hydroxylated fatty acid. Previously, the isolation of purgins I−III,9 partially acylated ester-type dimers of branched pentasaccharides from the jalap root, permitted the characterization of these compounds as individual macrolactones of distinctive glycosidic acids. Therefore, the hypothesis of a high molecular weight polymeric structure for this class of compounds had to be revisited. The structural complexity of the highly polar alcohol-soluble resin glycosides has seriously hampered the isolation of the individual glycolipids, limiting chemical studies to the characterization of their degradation products, i.e., esterifying organic acids and major glycosidic acids.2,10 Saponification of the jalap root MeOH-soluble contents afforded purgic acids A and B (1 and 2) as their major glycosidic acids.2 Compound 1 was identified as (11S)-hydroxytetradecanoic acid 11-O-β-D-quinovopyranosyl-(1→2)-O-β-D-glucopyranosyl-(1→3)-O-[β-D-fucopyranosyl-(1→4)]-O-α-L-rhamnopyranosyl-(1→2)-O-β-D-glucopyranosyl-(1→2)-O-β-D-quinovopyranoside. Compound 2

esin glycosides are complex mixtures of glycolipids found in members of the morning glory family (Convolvulaceae).1 Several biological effects of the crude extracts of the purgative Mexican jalaps (e.g., Ipomea purga, I. orizabensis, and I. stans) have been attributed to their resin glycosides.1,2 These resin glycosides represent noncytotoxic inhibitors of multidrug efflux pumps in Gram-positive3 and -negative4 bacteria as well as in mammalian cancer cells.5 Phytochemical reports on the jalap root (“officinal jalap”, I. purga), a drug used since pre-Columbian times in Mexico,2 were published as early as the second half of the 19th century.6 Most of the early and even recent botanical and chemical descriptions are confusing because of poor plant material identification. HPLC and 13C NMR spectroscopic profiles of the glycosidic acids obtained through saponification of the resin glycoside contents were used as analytical tools for authentication and quality control of the Mexican jalaps.2,7 The ratio of ether-soluble (“jalapin”) to alcohol-soluble (“convolvulin”) resin glycoside portions can also distinguish between authentic and false crude drugs; for example, the officinal jalap root yields the highest amount of MeOH-soluble resin glycosides (15−20% dried weight), in comparison to the yields from the Mexican scammony, or false jalap (I. orizabensis): 10−18% ether-soluble resins and 10 μg/mL) when tested against colon (HCT-15), cervix (HeLa), and breast (MCF-7 and MDA-MB-231) carcinoma cell lines (Tables 2 and S1, Supporting Information).15 Thus, its potential as an MDR modulator was explored. Results of the modulation assay are shown in Table 2, and this screening utilized both vinblastinesensitive and vinblastine-resistant human breast carcinoma cells C

DOI: 10.1021/np500762w J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 2. Modulation of Vinblastine Cytotoxicity in Drug-Sensitive MCF-7 and Multidrug-Resistant MCF-7/Vin by Jalapinoside (4) reversal foldc

IC50 (μg/mL) compounda

MCF-7/Vin−

MCF-7/Vin+

MCF-7 sens

RFMCF‑7/Vin−

RFMCF‑7/Vin+

RFMCF‑7 sens

vinblastine 4 reserpineb

1.02 ± 0.18 1906) indicated that jalapinoside (4) is extremely potent at 25 μg/mL, as previously observed for other morning glory resin glycosides, e.g., murucoidin V (RF 5a 10b which MCF‑7/Vin+ 255) and purgin II (RFMCF‑7/Vin+ >2140), are proven substrates of glycoprotein P. These results further support the potential of this family of compounds as inhibitors of multidrug efflux pumps in mammalian cancer cells, where glycoprotein-P is the chief plasma membrane-associated translocase responsible for the MDR phenotype,5a but also highlight their importance for overcoming MDR in cancer therapy.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points (uncorrected) were determined on a Fisher-Johns apparatus. Optical rotations were measured on a Perkin-Elmer 341 polarimeter. 1H (700 MHz) and 13C (175 MHz) NMR experiments were performed on a Bruker Avance III HD NMR spectrometer. Chemical shifts were referenced to TMS, and J values are given in Hz. ESIMS, HRESIMS, and MALDI-TOFMS were recorded on Bruker Daltonics Esquire 6000, Thermo LTQ Orbitrap XL, and Bruker MicrO-TOF-Q mass spectrometers, respectively. Waters HPLC equipment was composed of a 600E multisolvent delivery system with a refractive index detector (Waters 410). Control of the equipment, data acquisition, and processing of the chromatographic information was performed by the Empower 2 software (Waters). HPLC-ESIMS was carried out on an Agilent 1200 Rapid Resolution liquid chromatograph coupled to a Bruker Daltonics Esquire 6000 mass spectrometer. GC-MS was performed on a Thermo-Electron instrument coupled to a ThermoElectron spectrometer. GC conditions: DB-5MS (5% phenyl)methylpolysiloxane column (30 m × 0.25 mm, film thickness 0.18 μm); He, linear velocity 30 cm/s; 2 mL/min; 50 °C isothermal for 4 min, linear gradient to 300 °C at 40 °C/min; final temperature hold, 20 min. MS conditions: ionization energy, 70 eV; ion source temperature, 250 °C; interface temperature, 270 °C; scan speed, 2 scans s−1; mass range, 45−600 amu. TLC was carried out on precoated Macherey−Nagel silica gel/UV254 plates of 0.25 thickness, and spots were visualized by spraying with 3% CeSO4 in 2 N H2SO4 followed by heating. Chemicals, Cell Lines, and Cell Cultures. Colon (HCT-15), cervix (HeLa), and breast (MCF-7 and MDA-MB-231) carcinoma cell lines were obtained from the American Type Culture Collection. The resistant MCF-7/Vin was developed through continuous exposure to vinblastine during five consecutive years as previously reported.5a All cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and were cultured at 37 °C in an atmosphere of 5% CO2 in air (100% humidity). To maintain drug resistance, MCF-7/Vin+ cells were cultured in medium containing 0.192 μg/mL of vinblastine. At the same time, a stock of MCF-7/Vin− cells was maintained in vinblastine-free medium. Plant Material. The roots of I. purga (Wender) Hayne were collected in Coxmatla, Municipio de Xico, Veracruz, Mexico, in November 2010 and identified by botanist Alberto Linajes. Three voucher specimens were deposited at the following herbaria: Instituto D

DOI: 10.1021/np500762w J. Nat. Prod. XXXX, XXX, XXX−XXX

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min, linear gradient to 300 at 20 °C/min. Retention times for TMS derivatives of common sugars were used as standards for GC identification: D-fucose, tR 4.53; L-rhamnose, tR 4.58 min; D-quinovose, tR 4.64 min; and D-glucose, tR 6.53 min. The derivatized (CH2N2) organic layer residue obtained from acid-catalyzed hydrolysis was submitted to normal-phase HPLC (ISCO, 21.2 m × 250 mm, 10 μm) using an isocratic elution of hexanes−CHCl3−acetone (6:3:1)2 and a flow rate of 6 mL/min to give 0.3 mg of methyl (3S,11S)dihydroxytetradecanoate (ipurolic acid methyl ester): tR 20.8 min; colorless needles; mp 68−70 °C; [α]D +13 (c 0.02, MeOH); HPLCESIMS m/z 273 [M − H]−, 255 [273 − H2O]−, 231 [M − CH3(CH2)2]−, 103 [CH(OH)CH2COOCH3]−, 73 [CH3(CH2)2CH(OH)]−; identified by comparison of melting point and optical rotation with published values.12a The preparation and identification of 4-bromophenylacyl (2S)-2methylbutyrate were performed according to reported procedures:11 mp 40−41 °C; [α]D +16 (c 0.03, MeOH); GC-MS (tR 4.75 min) m/z [M + 2]+ 272 (6.8), [M]+ 270 (7.3), 254 (3.8), 252 (3.8), 186 (2.1), 172 (8.6), 171 (100), 70 (9.7), 169 (88.7), 90 (13.9), 89 (23.4), 85 (11.5), 63 (5.3) 57 (19), 51 (2.3), 50 (2.9), 41 (8.5), 39 (9.4). This transesterification procedure has been used to confirm the absolute configuration for 2-methylbutyric acid.16 Cytotoxicity and Modulation of Multidrug-Resistance Assays. The cytotoxicity and MDR reversal of compound 4 were determined by using the SRB assay.15 The cells were harvested at log phase of their growth cycle, treated in triplicate with various concentrations of the test samples (0.2−25 μg/mL), and incubated for 72 h at 37 °C in a humidified atmosphere of 5% CO2. The assay was carried out under static conditions, and the results are expressed as the concentration that inhibits 50% control growth after the incubation period (IC50). The values were estimated from a semilog plot of the drug concentration (μg/mL) against the percentage of growth inhibition.15 Vinblastine was included as a positive control. The reversal effects as modulators were further investigated with the same method. MCF-7 and MDR MCF-7/Vin cells were seeded into 96-well plates and treated with various concentrations of vinblastine (0.000 64−10 μg/mL) in the presence or absence of glycolipids (dissolved in a mixture of water−DMSO, 9:1) at 25 and 5 μg/mL for 72 h as previously described.5a The ability of glycolipids to potentiate vinblastine cytotoxicity was measured by calculating the IC50 as described above. In these experiments, reserpine (5 μg/mL) was used as a positive control drug. The reversal fold value, as a parameter of potency, was calculated from dividing IC50 of vinblastine alone by IC50 of vinblastine in the presence of test compounds.

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REFERENCES

(1) Pereda-Miranda, R.; Rosas-Ramírez, D.; Castañeda-Gómez, J. In Progress in the Chemistry of Organic Natural Products; Kinghorn, A. D., Falk, H., Kobayashi, J., Eds.; Springer-Verlag: New York, 2010; Vol. 92, Chapter 2, pp 77−152. (2) Pereda-Miranda, R.; Fragoso-Serrano, M.; Escalante-Sánchez, E.; Hernández-Carlos, B.; Linares, E.; Bye, R. J. Nat. Prod. 2006, 69, 1460−1466. (3) (a) Pereda-Miranda, R.; Kaatz, G. W.; Gibbons, S. J. Nat. Prod. 2006, 69, 406−409. (b) Chérigo, L.; Pereda-Miranda, R.; FragosoSerrano, M.; Jacobo-Herrera, N.; Kaatz, G. W.; Gibbons, S. J. Nat. Prod. 2008, 71, 1037−1045. (c) Chérigo, L.; Pereda-Miranda, R.; Gibbons, S. Phytochemistry 2009, 70, 222−227. (d) EscobedoMartínez, C.; Cruz-Morales, S.; Fragoso-Serrano, M.; Rahman, M. M.; Gibbons, S.; Pereda-Miranda, R. Phytochemistry 2010, 71, 1796− 1801. (4) (a) Corona-Castañeda, B.; Pereda-Miranda, R. Planta Med. 2012, 78, 128−131. (b) Corona-Castañeda, B.; Chérigo, L.; Fragoso-Serrano, M.; Gibbons, S.; Pereda-Miranda, R. Phytochemistry 2013, 95, 277− 283. (5) (a) Figueroa-González, G.; Jacobo-Herrera, N.; Zentella-Dehesa, A.; Pereda-Miranda, R. J. Nat. Prod. 2012, 75, 93−97. (b) CruzMorales, S.; Castañeda-Gómez, J.; Figueroa-González, G.; MendozaGarcía, A. M.; Lorence, A.; Pereda-Miranda, R. J. Nat. Prod. 2012, 75, 1603−1611. (6) Eich, E. Solanaceae and Convolvulaceae: Secondary Metabolites. Biosynthesis, Chemotaxonomy, Biological and Economic Significance (A Handbook); Springer: Heidelberg, 2008; pp 532−582. (7) The crude drug consists of the dried, usually fragmented, rhizomes. The ochre-yellow to dark brownish pieces often have a powdery surface, a characteristically faint smoky odor, and a somewhat acrid, sweetish taste. The Mexican jalaps have also been employed as an antihelmintic and galactogogue and in the treatment of abdominal fever, dysentery, epilepsy, hydrocephaly, skin spots, meningitis, and tumors. A decoction of the root of I. purga is normally prepared using a 2 cm section of root to 1 L of water. The usual recommendation is to drink one cup of the cold decoction before bedtime. The recommended dosages of jalap root (to 1 L of water) are 1−3 g if a powder, 0.2−0.4 g if an extract, 0.1−0.6 g if a resin, and 10 to 20 drops every 4 h if a tincture; if given in sugar or jelly, this remedy is a safe purge for children. A teaspoon of the root, cut small or granulated, to a cup of boiling water is suggested. (8) Mannich, C.; Schumann, P. Arch. Pharm. Ber. Dtsch. Pharm. Ges. 1938, 276, 211−226. (9) (a) Castañeda-Gómez, J.; Pereda-Miranda, R. J. Nat. Prod. 2011, 74, 1148−1153. (b) Castañeda-Gómez, J.; Figueroa-González, G.; Jacobo, N.; Pereda-Miranda, R. J. Nat. Prod. 2013, 76, 64−71. (10) Singh, S.; Stacey, B. E. Phytochemistry 1973, 12, 1701−1705. (11) Pereda-Miranda, R.; Hernández-Carlos, B. Tetrahedron 2002, 58, 3141−3154. (12) (a) Ono, M.; Takagi-Taki, Y.; Honda-Yamada, F.; Noda, N.; Miyahara, K. Chem. Pharm. Bull. 2010, 58, 666−672. (b) Ono, M.; Imao, M.; Miyahara, K. Chem. Pharm. Bull. 2010, 58, 1232−1235. (13) (a) Hara, S.; Hikaru, O.; Kunihide, M. Chem. Pharm. Bull. 1987, 35, 501−506. (b) Miyase, T.; Saitch, H.; Shiokawa, K.; Ueno, A. Chem. Pharm. Bull. 1995, 43, 466−472. (14) Pereda-Miranda, R.; Mata, R.; Anaya, A. L.; Wickramaratne, D. B. M.; Pezzuto, J. M.; Kinghorn, A. D. J. Nat. Prod. 1993, 56, 571−582. (15) (a) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1107−1112. (b) Vichai, V.; Kirtikara, K. Nat. Protoc. 2006, 1, 1112−1116. (16) Bah, M.; Chérigo, L.; Taketa, A. T. C.; Fragoso-Serrano, M.; Hammond, G. B.; Pereda-Miranda, R. J. Nat. Prod. 2007, 70, 1153− 1157.

ASSOCIATED CONTENT

* Supporting Information S

1D and 2D NMR spectra as well as the ESIMS/MS data of compound 4 are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +52-55-5622-5288. Fax: +52-55-5622-5329. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the financial support by Dirección General de Asuntos del Personal Académico, UNAM (IN212813), and Consejo Nacional de Ciencia y Tecnologiá (220535). E.B. is grateful to DGAPA for a postdoctoral scholarship. Thanks are ́ due to G. Duarte (Facultad de Quimica, UNAM) and C. ́ Márquez (Instituto de Quimica, UNAM) for the recording of mass spectra. E

DOI: 10.1021/np500762w J. Nat. Prod. XXXX, XXX, XXX−XXX